Placement of the Angelina within and observations of Angelina rufescens

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Citation Karakehian, J. M., K. F. LoBuglio, and D. H. Pfister. 2014. “Placement of the Genus Angelina Within Rhytismatales and Observations of Angelina Rufescens.” Mycologia 106 (1) (January 1): 154–162. doi:10.3852/13-174.

Published Version doi:10.3852/13-174

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:30167530

Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Mycologia, 106(1), 2014, pp. 154–162. DOI: 10.3852/13-174 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897

Placement of the genus Angelina within Rhytismatales and observations of Angelina rufescens

Jason M. Karakehian1 Discomyceteae exsiccatae as No. 16 (Korf 1954). He Katherine F. LoBuglio referred us to a classic paper on this species by Donald H. Pfister Durand (1902), which Korf also cited in his treatment Farlow Herbarium of Harvard University, 22 Divinity of discomycetes (Korf 1973). Subsequent morpholog- Avenue, Cambridge, Massachusetts 02138 ical and sequence analyses resolved the placement of this genus not in the , where Durand (1902) and Korf (1973) had placed it, but instead Abstract: Angelina rufescens is placed within the core among the core clade of Rhytismatales (Lantz et al. clade of Rhytismatales (, Pezizomyco- 2011). tina, ) based on analysis of LSU and mtSSU rDNA. The only species in the genus, it MATERIALS AND METHODS produces distinctive ascomata that reoccur annually on wood and on the remains of its own previous Collection.—Photographs were taken in situ and collections fructifications, forming dense conglomerations of taken for cultural and morphological studies and molecular interlocking longitudinally elongated apothecia with sequencing. The remaining portions were dried at low gray hymenia. Known collections and references of A. temperature in a home food dehydrator and accessioned rufescens indicate that it is endemic to eastern and into FH (Karakehian 12040101). central United States. Morphological and cultural Culture.—A few hours after gathering A. rufescens, dis- characters are described with notes on ascomata charged spores were collected onto Difco PDA media in two development. No mitospores were observed in field 100 mm Petri dishes. Small sections of the substratum collections or in culture. Lectotypes are designated supporting 2–3 apothecia were removed from the collection for Hysterium rufescens and its synonym Ascobolus and placed on moistened filter paper elevated on four glass conglomeratus. Angelina rufescens is illustrated here for slides. Plates were rotated 90 degrees every 20 min, and the first time in the taxonomic literature. three regions of ejected ascospores were collected. The Key words: , lectotypification, Leotio- dishes were placed in a dark incubator at 23 C. Polysporous mycetes, Lewis David von Schweinitz, , subcultures were made by excising 2 mm cubes of spore- bearing media and placing them at multiple points in mitochondrial SSU, Nils Peter Angelin, nuclear LSU, 100 mm dishes of Difco PDA, which were maintained at 23 C ontogeny, in darkness.

Morphological observations.—Crush mounts from the living INTRODUCTION ascomata of A. rufescens were made in tap water or in 3% A collection of Angelina rufescens (Schwein.) Duby KOH and stained with phloxine in water and Congo red in was made in Carlisle, Massachusetts, United States 1 ammonia. Dried material was rehydrated in sealed Petri Apr 2012 from the underside of an oak log. The dishes on water-dampened filter paper, transferred to had been observed at least 3 mo and had dilute gum arabic for 12 h and sectioned to 20 mmona freezing microtome. Dried material for crush mounts was fruited yearly on the same log since it first was rehydrated in 3% KOH or dipped in 70% ethanol, discovered five years earlier. Initially the identification rehydrated in water and stained with Congo red. Ascospore of this fungus even to the family level proved elusive. sizes are given from the average of 30 spores discharged It seemed to possess characters that were reminiscent from living apothecia onto a cover slip and mounted in of genera in both the Dermateaceae and Rhytismata- water. India ink was employed to detect the presence of ceae, which we could not reconcile. We decided to gelatinous spore sheaths. Observations of ascomatal devel- sequence it, and at the same time Richard P. Korf opment were made from living field-collected material identified the fungus as A. rufescens based on other (Karakehian 12090801). Microscopic observations were material from New England provided by our col- made with Nomarski and brightfield optics on Olympus league Lawrence Millman (Millman 2012). Korf BH-2 and Olympus BX40 compound microscopes and an noted that he had seen this fungus many years earlier Olympus SZX9 stereomicroscope. Drawings were made by JMK with a Leitz Laborlux compound microscope fitted around Ithaca, New York, and had issued it in his with a Leitz drawing tube.

Submitted 27 May 2013; accepted for publication 20 Sep 2013. DNA Isolation, PCR and sequencing techniques.—DNA 1 Corresponding author. E-mail: [email protected] samples were obtained from living fruiting bodies of A.

154 KARAKEHIAN ET AL.: ANGELINA RUFESCENS (RHYTISMATALES) 155 rufescens (Karakehian 12040101). The DNA sample was Observations of ascomata development.—Hemispheri- used in PCR amplification and sequencing of the LSU and cal primordia, 100–150 mm diam, develop superficially mtSSU rDNA regions. DNA was extracted with the Qiagen on the substratum. These are at first hyaline but soon DNeasy Plant Mini Kit (cat. No. 69104). A 1/10 and 1/100 darken to pale yellow, orange then red with their apices dilution of the DNA was used for PCR amplification of the appearing granular, then tuberculate, from protruding mtSSU and LSU rDNA (ribosomal DNA) regions. The mtSSU was amplified with primers mrSSU1 and mrSSU3r cells (FIG. 2a, b). The primordia enlarge as hyaline (Zoller et al. 1999). Amplification of the LSU rDNA region hyphae emerge from their flanks and extend down, used primers LROR and LR5 (Monclavo et al. 2000). PCR infiltrating crevices in the surrounding substratum amplification, purification and sequencing was as described (FIG. 2c). These hyphae are septate, 3.5–5.5 mmdiam in Hansen et al. (2005). The GenBank accession numbers and sparsely encrusted with crystalline material. The for the LSU and mtSSU sequences for this isolate of A. primordia begin to elongate and enlarge laterally, rufescens are JX624162 and JX624163. An independent becoming hysterioid and darkening to dark brown- DNA extraction was performed from the living cultures black. Shallow apical sulci develop along their axes. The established in this study (see above). LSU and MtSSU DNA tissues surrounding the sulci and at the elongating ends sequences were determined to confirm the identity of the culture as A. rufescens. remain red-brown (FIG. 2d). Along the flanks of the primordia subtle longitudinal striations develop and Sequence analysis.—A NCBI (http://www.ncbi.nlm.nih. the hyphal extensions collapse forming a loose, flaky gov/) BLAST query of the LSU and mtSSU rDNA region pale brown tissue and leaving a white tomentose halo of determined that A. rufescens was most similar to genera in hyphal extensions only at the elongating ends (FIG. 2e). the Rhytismatales. LSU and mtSSU rDNA sequences from In transverse section the sulci are composed of 69 taxa representing members of the Rhytismatales sensu involute filamentous hyphae of the developing stricto (Lantz et al. 2011) were included in a combined apothecial margins. Crystaline material accumulates phylogenetic analyses of the LSU and mtSSU rDNA region. around the hyphae, ultimately forming a distinct split Phacidium lacerum, Lachnum bicolor and Pezicula carpinea (FIG. 2f). A thin pruinose layer of this material can be were used as outgroup taxa. These taxa and their GenBank accession numbers are in Lantz et al. (2011). observed developing around the sulci on the outer Alignment of DNA sequences was done with Clustal W surface of the primordia. through the Cipres Science Gateway (Miller et al. 2009) and Ascospores mature before the opening of the manually adjusted with Se-Al 2.0a8 (Rambaut 1996). The apothecium. In moist conditions, the prosenchyma- combined LSU and MtSSU DNA alignment is available from tous regions of the excipula swell, pushing the involute TreeBase under S14647. Regions of the DNA sequences that margins up and outward. Hymenial elements expand were highly divergent, as described by Lantz et al. (2011), and asci extend upward through the hamathecia, were not included in the analysis and resulted in a dataset of pushing the apothecium open further. Where asco- 1804 base pairs. DNA sequence alignments were analyzed as described by LoBuglio and Pfister (2010) with: MrBayes mata are densely gregarious, the flanks of the adjoining 3.0b4 (Ronquist and Heulsenbeck 2003) for obtaining apothecia swell tightly against each other, thus appear- Bayesian posterior probabilities (PP); maximum parsimony ing as interlocking gray hymenia. In senescent apothe- (MP) with PAUP 4.0b10 (Swofford 2002); and maximum cia where the excipula are heavily carbonized, the likelihood (ML) with RAxML-HPC2 on Abe through the apothecia tend to lose the ability to close. Cipres Science Gateway (Miller et al. 2009). Branch support for MP and ML analyses was determined by 1000 bootstrap replicates. Angelina Fr., Summa veg. Scand., 2:358. 1849. RESULTS TYPE SPECIES: Ascobolus conglomeratus Schwein. Molecular phylogenetic analyses.—Results from phylo- genetic analyses of the combined LSU and mtSSU Angelina rufescens (Schwein.) Duby, Me´m. Soc. Phys. DNA sequences of 69 taxa, including A. rufescens, are Gene`ve 16:51. 1861. ; Hysterium rufescens Schwein., Schriften Naturf. Ges. presented (FIG. 1). Parsimony analysis produced 3441 Leipzig 1:50. 1822. most parsimonious trees 2491 long (504 sites were parsimony informative, 173 were parsimony uninfor- 5 Ascobolus conglomeratus Schwein., Trans. Amer. Philos. mative and 1127 sites were constant). Results from Soc., n.s., 4:178. 1832. ML, MP and Bayesian analyses placed A. rufescens with ; Angelina conglomerata (Schwein.) Fr., Nova Acta Regiae high support (95, 79, 100 ML, MP and Bayesian Soc. Sci. Upsal., ser. 3, 1:121. 1851. analyses respectively) as sister to all other taxa in a Lectotypes designated here: Hysterium rufescens clade containing predominately species of Hypoderma Schwein. (1822) PH, envelope No. 01104792, bar and Lophodermium. code No. 00064695, ex Collins Collection, packet 156 MYCOLOGIA

FIG. 1. Phylogenetic placement of Angelina rufescens among representatives of the Rhytismatales. Maximum likelihood tree from analysis of LSU and mtSSU DNA sequences. Maximum likelihood and maximum parsimony bootstrap values above 70% are given above the branches and are separated by a forward slash. Bayesian PP, values above 90% are below the branches. The numbers following genera with identical species have collection numbers following name. The GenBank accession numbers for the LSU and mtSSU sequences for this isolate of A. rufescens are JX624162 and JX624163. GenBank accession numbers of all other isolates in the phylogenetic tree are included in Lantz et al. 2011.

No. 11 ‘‘Beth’’ [Bethlehem, Pennsylvania]; Ascobolus of deciduous trees, particularly Castanea dentata and conglomeratus Schwein. (1832) PH, envelope Number Quercus spp. The affected wood is reduced to a soft, 01102746, bar code Number 00062099, ‘‘Beth’’ [Bethle- fibrous state, creating a texture that appears fleshy hem, Pennsylvania]. and creamy white from mycelium and deposited Etymology of the generic name: In honor of the Swedish crystalline material. Regions adjacent to the affected paleontologist and naturalist Nils Peter Angelin (1805– area remain hard and intact. At low magnification, 1876) (Fries 1849, Anonymous 1876). dense white mats of appressed hyphae frequently are Range: Maine south to northern Alabama and observed in the crevices of the wood or covering the Georgia; Massachusetts west to Iowa. dead, persistent remains of previous fruitings of A. Seasonality: Ascomata with mature ascospores are rufescens. Apothecia develop on bare wood or upon found from October through May. layers of these remains, which after multiple seasons Substratum: Surfaces of decorticated tree stumps of fruiting and senescence in older colonies, accu- and the cut ends and undersides of logs and branches mulate into a spongy, heterogeneous layer of fungal KARAKEHIAN ET AL.: ANGELINA RUFESCENS (RHYTISMATALES) 157

FIG. 2. Progression of ascomatal development of A. rufescens (Karakehian 12090801). a. Young hemispherical primordia (arrows). b. Primordia enlarging and darkening. c. Primordia with hyaline hyphal extensions. d. Hysteriform primordia with elongating ends composed of pale brown tissue with hyphal extensions. e. Hyphal extensions surrounding the ends of primordia as they elongate over the surrounding substratum. f. Transverse section of a primordium shown in e. Bars 5 100 mm. tissue, debris and crystalline material over the basal Excipulum. In transverse section 65–100 mm thick, woody substratum. stromatic, composed of prosenchymatous and pseu- Ascomata, macromorphological features. Sessile, su- doparenchymatous regions with deposited extracellu- perficial, elongated apothecial disks, flexuous, genic- lar pigments (FIGS. 4a–ii, 5a). The pseudoparenchy- ulate or trilaterally branched, never confluent, matous region is composed of indistinct isodiametric scattered or gregarious and appearing in mass as or interwoven elongate cells, golden brown, gray- densely packed expanses of interlocking gray hyme- brown or dark brown with a highly carbonized central nial surfaces each bordered by white to brown region (FIGS. 4a–iia, 5c). Nearly the entire excipulum margins, forming effuse masses several cm to dm in is carbonized in older apothecia. A prosenchymatous extent (FIG. 3a, b). Single ascomata 1–3 mm long 3 region, 15–26 mm thick is adjacent to the hymenium, 0.5–1 mm wide 3 0.4–0.6 mm thick. Hymenia flat, composed of parallel, elongate, pale brown cells that concave to slightly convex, slate gray, granular from originate in the intra-stromal matrix beneath the asci projecting beyond the hymenial surface subhymenium and extend into the margin (FIGS.4a– (FIG. 3c). Margins even with, or barely projecting iib, 5d). above, the hymenium; fibrous, with a laciniate edge, Intra-stromal matrix (after Sherwood, 1980). Located white to pale cream to brown. Flanks stromatic, below the subhymenium and arising from the excipu- black, fleshy or somewhat coriaceous, smooth or lum, 60–150 mm thick, composed of hyaline pseudo- longitudinally striate, sometimes encrusted with pale parenchyma with discrete pockets of gray-brown brown tissue. In dry conditions, the margins roll extracellular pigment, becoming prosenchymatous as inward and the apothecia become nearly hysteriform cells form the subhymenium, in older apothecia much (FIG.3d). reduced (FIGS. 4a–iii, 5c). Apothecial margins. In transverse section 50–85 mm Subhymenium. Composed of densely packed, hya- thick, composed of hyaline-tipped filamentous hyphae line, cuboidal, globular and curling ascogenous originating within the excipulum, interspersed in a hyphae (FIG.4a–iv). crystalline matrix composed of irregularly shaped Hymenium. 160–200 mmthick(FIGS. 4a–v, 5a). granules and delicate fan-shaped fascicles of acicular Paraphyses. 1.5–2.5 mm wide, smooth, filiform, crystals that disappear in KOH (FIGS. 4a–i, 5b). In branching, septate from the base to the middle, the older apothecia, the hyphae become darkened at the tips straight, sinuous or curled, interwoven to form an tips and the crystalline material may wear away, epihymenium, anastomosing near the bases to form H- resulting in brown and eroded margins. shaped hyphal bridges (FIG.4b). 158 MYCOLOGIA

FIG.3. Angelina rufescens (a. Karakehian 12111102; b–d. Karakehian 12040101). a. Macroscopic view of the habit of ascomata. Bar 5 5 cm. b. Ascomata (note dead persistent ascomata of previous fruitings of A. rufescens in the upper center of the image). Bar 5 1 mm. c. Granular surface of hymenia. Bar 5 1 mm. d. Dry ascomata. Bar 5 1 mm.

Asci. Arising from croziers, clavate, thin-walled with Associated sterile structures. Solitary red bodies ap- a long stipe, 99–196 mm long from the base to the proximately 100 mm diam and adorned with short apex, 8–9 mm wide, apex acute, apical wall undifferen- hyphal appendages are observed in many collections tiated, no reaction with IKI or Meltzer’s reagent, scattered on the substrata among mature ascomata dehiscence via a pore-like apical rupture. Asci develop (FIG. 6a). These are most easily observed on decorti- sequentially. At maturity, apices project beyond the cated wood but are obscured within regions of dead surface of the hymenium. Eight-spored, biseriate near apothecia and debris. In transverse section they are the apex and uniseriate toward the base with the composed of a central region of densely packed lowest few ascospores running singly down into the hyaline plectenchyma surrounded by a rind of stipe (FIG.4c). parallel-oriented pale brown hyphae (FIG.6b).No Ascospores. Hyaline, smooth, without gelatinous spores were found associated with these structures. sheaths. Spores observed within asci are dimorphic Conidiomata. Not observed. with four aseptate spores nearest to the apex followed Culture. Ascospores germinated within 12 h by four one-septate spores. Aseptate ascospores cylin- (FIG. 4e). At 2 wk colonies 2–3 mm diam and white; drical with blunt ends, straight or slightly curved, the center tinged pink in reverse. Colonies appeared thin-walled, with refractive bodies at the poles, (14.1–) tomentose from dense growths of aerial hyphae, while 16.7(–19.7) 3 (3.8–)4.4(–5.6) mm. One-septate asco- the margins remained immersed in the media. Some spores cylindrical-fusoid, occasionally clavate with one hyphae contained small inclusions of red pigment. At cell attenuated, straight, relatively thicker-walled and 4 wk colonies were 6–10 mm diam, the pink cast in the slightly smaller than their aseptate counterparts at media intensified to a pale red-orange or peach and (11.3–)15.1(–18.8) 3 (2.8–)3.5(–4.7) mm, septum me- became vivid purple-pink with some rust-brown spots dian, not constricted or occasionally slightly so, refrac- at 7 wk (FIG. 7a). The margins remained white, and tive bodies present at the poles of each cell (FIG.4d). the hyphae in pigmented regions coalesced into short, KARAKEHIAN ET AL.: ANGELINA RUFESCENS (RHYTISMATALES) 159

of pale brown hyphae (FIG. 7b). Morphologically they are similar to the red hemispherical bodies observed in natural collections. No spores were found associated with these structures. At 17 wk no further differenti- ation of tissues had occurred and pigmentation had not changed. The globose bodies continued to develop singly or in small groups. The fungus deposited noticeable amounts of crystalline material into the media. This was composed of irregularly shaped granules, fascicles of oblong granules and delicate feather-like fascicles of acicular granules (FIG. 8a–c). Nine months later, the largest colony was 2.5 cm diam and growth had stalled. Lowering the temperature to 18 C had little effect on the rate of growth compared with colonies kept at room temper- ature. Samples from cultures were taken for sequenc- ing and it was observed that the colonies were tough, rubbery and difficult to cut with a scalpel. Specimens examined: UNITED STATES. CONNECTICUT: West Haven, on Castanea trunk, Oct 1888, R. Thaxter 39 (FH). MAINE: Kittery, Oct 1885, R. Thaxter (FH). Kittery Point, Oct & Nov 1885, R. Thaxter 4175 (FH). Dodge Point, Newcastle, on dead wood, 9 Sep 1995, S. Ristich (FH). MASSACHUSETTS: Canton, on chestnut stump, May 1932, D.H. Linder (FH). Carlisle, on Quercus log, 1 Apr 2012, J.M. Karakehian 12040101 (FH 00290519). Carlisle, on Quercus log, 8 Sep 2012, J.M. Karakehian 12090801 (FH 00290521). Blackstone, on fallen Quercus log, 11 Nov 2012, J.M. Karakehian 12111102 (FH 00290520). Sandwich, on branch of Quercus sp. on the ground, 11 Apr 1937, G.D. Darker FIG. 4. Ascomatal structures and germinated ascospores 6327 (FH). NEW JERSEY: On Quercus stump, Jan 1880, J.B. of A. rufescens (Karakehian 12040101). a. Diagram of Ellis 466, North American Fungi (FH). NEW YORK: apothecial tissues in transverse section with each region Gannett Hill, on end of stumps, 11 May 1947, C.T. Rogerson labeled i–v: i. margins; ii. excipulum; iia. pseudoparenchy- 1435 (FH). McGowan’s Woods, Ithaca, on stump, 18 Apr matous region of the excipulum; iib. prosenchymatous 1903, H.S. Jackson 307 (FH). McGowan’s Woods, Ithaca, region of the excipulum; iii. internal stroma; iv. subhyme- end of rotten log, 8 Oct 1902, E.J. Durand 1777 (FH). 5 m 5 nium; v. hymenium. Bar 250 m. b. Paraphyses. Bar Ringwood Swamp, Ithaca, on top of chestnut stump, 14 Nov 10 mm. c. Asci. (right to left) two mature asci, ascus after 1936, W.L. White & B.L. Richards 2761 (FH). Ringwood, 30 5 spore discharge, ascus tip during spore discharge. Bar Apr 1934, W.W. Ray 2418 (FH). PENNSYLVANIA: Bethle- m 5 m 10 m. d. Ascospores. Bar 10 m. e. Ascospores in various hem, L.D. von Schweinitz, packet 11 (Hysterium rufescens, stages of germination (in lactophenol/cotton blue). Bar 5 PH–LECTOTYPE, envelope 01104792, bar code 00064695, 50 mm. ex Collins Collection). Bethlehem, L.D. von Schweinitz (Hysterium rufescens, PH envelope 01104791, bar code acicular, hyphal cords. In reverse the colonies were a 00064694). Bethlehem, L.D. von Schweinitz (Ascobolus dingy brown-pink. At this time three light and conglomeratus, PH– LECTOTYPE, envelope 01102746, bar temperature regimes were established: i) 18 C and a code 00062099). VERMONT: Lake Dunmore, 1896, W.G. light/dark of 12 h with a 10 watt CFL bulb, ii) total Farlow, herbarium of the New England Botanical Club darkness, iii) room temperature in diffuse daylight. In (FH). WEST VIRGINIA: Ronceverte, 1 Jan 1881, C.G. week 10, small regions near the margins in colonies Pringle 1258 (FH). exposed to light developed dense growth of mostly confluent red-brown globose bodies. Colonies kept in DISCUSSION darkness did not develop these bodies. These bodies were approx. 150 mm and bore short hyphal cords at Previous treatments.—Durand (1902) provides an their apices or a sparse growth of short hyphae excellent and concise account of the taxonomic covering the entire body. In transverse section they history of A. rufescens. We add only in regard to the were composed of an internal region of densely synonym Angelina conglomerata, the combination of interwoven hyaline plectenchyma with an outer rind which was not made by Fries when he erected the 160 MYCOLOGIA

FIG. 5. Apothecial tissues of A. rufescens (Karakehian 12040101) in transverse section; 20 mm sections in Congo red. a. Apothecium. Bar 5 200 mm. b. Margin. Bar 5 50 mm. c. Same apothecium showing pseudoparenchymatous tissue of the excipulum and detail of adjoining intra-stromal matrix. Bar 5 100 mm. d. The same apothecium showing prosenchymatous tissue of the excipulum. Bar 5 100 mm. genus Angelina in 1849 but in a subsequent study (Fries 1851).

Phylogeny.—In their phylogenetic analysis of Rhytis- matales, Lantz et al. (2011) outlined a core clade that comprised mostly taxa of Rhytismataceae, Cudonia- ceae and maximus (Fr.) Rehm. Character mapping was employed, and the core clade was divided further into two strongly supported clades according to the plane of symmetry of the ascomata. To facilitate discussion in their study, informal designations of radiate and bilateral were assigned to the two clades. These terms describe taxa that possess circular ascomata that open in a radiate FIG. 6. Sterile red bodies associated with ascomata of fashion, such as spp., and taxa in which Angelina rufescens on the surface of substratum (living) the ascomata are elliptical or oblong and open along (Karakehian 12040101). a. Detail of two sterile bodies in a single slit, such as those in Lophodermium and profile. Bar 5 100 mm. b. Transverse section of sterile body Hypoderma (Lantz et al. 2011). In our analysis, with encrusted hyphal extensions on the upper surface. Bar Angelina rufescens occupies its own branch within 5 200 mm. KARAKEHIAN ET AL.: ANGELINA RUFESCENS (RHYTISMATALES) 161

FIG. 8. Crystaline structures deposited into media surrounding colonies of A. rufescens. a. Irregularly shaped granules. b. Fascicles of oblong granules. c. Feather-like FIG. 7. Characteristics of Angelina rufescens in culture. a. Colony on PDA. Bar 5 10 mm. b. Transverse section of granules. Bars 5 50 mm. sterile bodies in culture. Bar 5 50 mm. colored; although it may be conspicuous when fruiting in effuse masses. When immature it superfi- the bilateral clade. Although its ascomata share cially resembles hysterioid genera of the Dothideo- important morphological characters with other taxa mycetes, and when mature it may be confused with in the core clade, such as opening along a single other apothecioid genera such as Mollisia. Until longitudinal slit, having undifferentiated, thin-walled recently there have been no illustrations in any ascus apices and hyaline ascospores, other characters popular or taxonomic forum. However, once the diverge in varying degrees from those traditionally fungus is correctly identified and the host substrata considered to circumscribe Rhytismataceae. Most noted, it may be instantly recognizable in the field if notable are a superficial habit, elliptical ascospores encountered again or deliberately searched for. that lack gelatinous sheaths and a cup-shaped Furthermore, because the fungus fruits year after excipulum; there is no discrete clypeal and basal year on the same substratum, that location might be layer as observed in Lophodermium and Hypoderma. revisited for further observations and collections. Only in the earliest developmental stages do the primordia exhibit a complete peridium of pigmented Distribution.—The collections and literature we ex- hyphae. These tissues soon differentiate into the amined indicate that the distribution of A. rufescens is distinctive involute margins of the closed, immature restricted to eastern and central USA in the temper- apothecia—a preformed zone of dehiscence instead ate northern hemisphere. Johnston (1997, 2001) of an irregular splitting of covering layer tissues. notes that the order in the southern hemisphere is There is no reduced excipulum lining the interior of probably as diverse genetically, morphologically and the apothecia; ascogenous elements emerge out of biologically as it is in the northern hemisphere, with the intra-stromal matrix within the centrum, and the saprophytic, parasitic and endophytic species present. hymenium is directly adjacent to the parallel oriented However, he notes that lignicolous taxa are more prosenchymatous tissue of the excipulum. These diverse in the northern hemisphere and less so in differences, along with those that are more conspic- Australasia, where there is a greater diversity of leaf- uous in members of the , further inhabiting species. Furthermore, few species are elucidate the diversity of ascomatal morphology shared between the two hemispheres (Johnston within Rhytismataceae. 2001). Most species of the Rhytismatales are restricted geographically, by host preference of a single species, Occurrence.—We compiled a list of approximately 50 genus or family and by tissue preference; that is leaves collections of A. rufescens. These are deposited at or wood (Johnston 1997). The fact that Quercus and various herbaria dating to ca. 1822. With the Castanea are northern hemisphere taxa argue for a exception of a few gaps of about 20 and 30 y, A. geographical restriction. A search of the literature for rufescens has been collected consistently but not temperate Asian collections was fruitless. frequently. The demise of the American chestnut in the eastern states caused by the chestnut blight fungus Cryphonectria parasitica (Murrill) M.E. Barr ACKNOWLEDGMENTS may be partly to blame. The fact that fruiting occurs Kanchi N. Gandhi for advising us on nomenclature and the through the winter when few collectors are afield may staff at the Harvard University Botany Libraries provided provide a further reason for the seeming rarity. necessary assistance. Richard Korf, Lawrence Millman and Furthermore, the fungus is discrete and not brightly Kathie Hodge helped solve the identity of this fungus in one 162 MYCOLOGIA way or another. Emma Williams at the Academy of Natural Lantz H, Johnston PR, Park D, Minter DW. 2011. Molecular Sciences of Drexel University, Philadelphia, Pennsylvania phylogeny reveals a core clade of Rhystismatales. (PH), facilitated the loans of the Schweinitz specimens. Simona Mycologia 103:57–74, doi:10.3852/10-060 Margaritescu at the Royal Ontario Museum, ROM Fungarium, LoBuglio KF, Pfister DH. 2010. Placement of Medeolaria Canada (TRTC), and Scott Redhead at the National Mycolog- Farlowii in the Leotiomycetes, and comments on ical Herbarium, Canada (DAOM), supplied information on sampling within the class. Mycol Prog 9:361–368, Canadian holdings of A. rufescens. Kay Fairweather for bringing doi:10.1007/s11557-009-0644-y this interesting fungus to our attention and for her persistence, MillerMA,HolderMT,VosR,MidfordPE,LiebowitzT,Chan over 5 y, in seeking a name for her find. L, Hoover P, Warnow T. The CIPRES Portals. http: //www. phylo.org/sub_sections/portal. 2009-08-04. (Archived by WebCiter at http: //www.webcitation.org/5imQlJeQa) LITERATURE CITED Millman L. 2012. Welcome back Angelina. Fungi 5:51. Anonymous. 1876. Professor Angelin, Phil. Doc. Geological Moncalvo JM, Lutzoni FM, Rehner SA, Johnson J, Vilgalys R. 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